JP2008501043A - Adhesive beads for immobilizing biomolecules and methods for producing biochips using the same - Google Patents

Abstract

The present invention relates to a bead for immobilizing biomolecules and a method for producing a biochip using the same, and more particularly, an adhesive bead having characteristics as an adhesive to the support for immobilizing the biomolecule and the surface of the chip substrate at the same time. And a method for producing a biochip, characterized in that a biomolecule is bound to the adhesive bead and an aqueous suspension of the bead on which the biomolecule is immobilized is produced and then immobilized on a substrate. is there.
Since the adhesive beads according to the present invention simultaneously have a feature as a support for fixing biomolecules and an adhesive to the surface of the chip substrate, they can be directly fixed on the biochip without a separate apparatus and treatment process.

Description

  The present invention relates to a bead for immobilizing biomolecules and a method for producing a biochip using the same, and more particularly, an adhesive bead having characteristics as an adhesive to the support for immobilizing the biomolecule and the surface of the chip substrate at the same time. Further, the present invention relates to a biochip manufacturing method, wherein a biomolecule is bound to the adhesive beads, an aqueous suspension of the beads having the biomolecule fixed thereon is manufactured, and then this is fixed on a substrate.

  Stationary phase supports capable of immobilizing biomolecules are widely used in many biological application fields that utilize selective affinity between biomolecules, typically agarose, cellulose, There are natural supports such as porous glass, silica, alumina and zeolite, and synthetic supports such as polyacrylamide beads, polymethacrylic acid beads, polystyrene beads and membranes (Regnier, FE, J. Chromatogr. Sci ., 14: 316, 1976; Hjerten, S., Anal. Biochem., 3: 109, 1962).

  In addition to traditional fields such as protein separation / purification or affinity chromatography, such stationary phase supports can be used in biochips used for high-throughput screening (HTS) and diagnostic applications ( Biochip) is widely used as a carrier for immobilizing biomolecules on chip substrates (Sato, K., Adv. Drug Deliv. Rev., 55: 379, 2003; Andersoon, H., Electrophoresis, 22: 249, 2001; Choi, JW, Biomed. Microdevices, 3: 191, 2001). This solves the technical limitations of the two-dimensional fixation method such as self-assembly that has been used in the field, that is, the problem that it is difficult to integrate biomolecules and preserve bioactivity. It has been proposed as an alternative for this. Due to the presence of the stationary phase support, biomolecules are supported at a high concentration using a large three-dimensional surface area, and this is immobilized on a chip substrate, thereby enabling biocompatible integration of biomolecules. .

  There are various forms of stationary phase supports that can be used. For example, the membrane form is a support utilizing a wide surface having a characteristic porous structure such as cellulose as a fixing surface for biomolecules, and the polymer matrix form is glucose, polylysine, chitosan, dextran on a substrate. This is a support in which a thin polymer matrix made of a biocompatible polymer such as polyallylamine and polyvinyl alcohol is formed to widen the immobilization surface and to improve the steric hindrance of the biomolecule to the substrate (Korea Patent Publication No. 2004). No. 0004725; Yakovleva, J., Biosens. Bioelectron., 19:21, 2003; Gill, I., Trends in Biotechnology, 18: 282, 2000; US 5,034,428; US 5,482,996).

  On the other hand, a bead-shaped support is an immobilization support that fixes biomolecules to individual beads having a spherical shape and collects them to form a three-dimensional structure having a large surface area. When formed, it can be used as a biochip (Sato, K., Adv. Drug Deliv. Rev., 55: 379, 2003); Andersoon, H., Electrophoresis, 22: 249, 2001; Choi, JW , Biomed. Microdevices, 3: 191, 2001). The membrane or polymer matrix support can be attached to the external environment when the immobilization of biomolecules is restricted around the surface in contact with the exterior or covalently bonded to enhance the immobilization force of the biomolecules. It has several disadvantages that it is difficult to maintain the physiological activity of sensitive enzymes or other proteins. Contrary to this, since the bead support is made of individual beads to which biomolecules are already fixed, a three-dimensional structure is made, and therefore, the surface area utilization ratio is high, and various immobilization methods that maintain the activity of biomolecules are used. The advantage is great because it can be utilized. In particular, it is very easy to handle, so it is easy to manufacture chips in the biochip manufacturing process where biomolecules must be fixed in microchannels, such as Lab-on-a-chip. It becomes a suitable material.

  Further, since existing beads themselves do not have adhesiveness to the chip substrate, there is a disadvantage that a separate method for fixing the beads in the fine flow path is necessary. Typical methods for immobilizing beads that have been developed and used to date include confining beads in a microchannel using a physical barrier, immobilizing using a magnetic field, and ultrasonic or laser tweezers. There is a method using (laser tweezer). However, these methods have the disadvantage that the selection of beads is limited, the processing steps for manufacturing the chip are complicated, the noise during optical measurement is large, and a separate device is required inside or outside the chip. Have. Therefore, there is a problem that it is somewhat uneconomical to apply as a lab-on-chip purpose (Sato, K., Adv. Drug Deliv. Rev., 55: 379, 2003; Andersoon, H., (Electrophoresis, 22: 249, 2001; Choi, JW, Biomed.Microdevices, 3: 191, 2001; Meng, A., Transducers, Sendai, Japan, 876, 1999; Dorre, K., Bioimaging, 5: 139, 1997) .

  On the other hand, the present applicant has filed a patent application (Korean Patent Application No. 10-2004) for a method of manufacturing a biochip characterized in that a probe or a bead to which the probe is fixed is fixed to the surface of a substrate using an adhesive. 104944). According to the patent, it is possible to fix a bead on which a probe is fixed to a substrate with an adhesive regardless of a physical barrier or an electric field. Still has the inconvenience of having to add a separate adhesive.

Therefore, the industry simultaneously has a feature as a support for immobilizing biomolecules and an adhesive to the surface of the chip substrate, and can be directly immobilized on the biochip without a separate apparatus and processing process. The development of sticky beads and biochips using such beads is eagerly desired.
Korean Patent Publication No. 2004-0004725 US 5,034,428 US 5,482,996 Regnier, FE, J. Chromatogr. Sci., 14: 316, 1976 Hjerten, S., Anal. Biochem., 3: 109, 1962 Sato, K., Adv. Drug Deliv. Rev., 55: 379, 2003 Andersoon, H., Electrophoresis, 22: 249, 2001 Choi, JW, Biomed. Microdevices, 3: 191, 2001 Yakovleva, J., Biosens. Bioelectron., 19:21, 2003 Gill, I., Trends in Biotechnology, 18: 282, 2000 (Sato, K., Adv. Drug Deliv. Rev., 55: 379, 2003) Andersoon, H., Electrophoresis, 22: 249, 2001 Choi, JW, Biomed. Microdevices, 3: 191, 2001 Meng, A., Transducers, Sendai, Japan, 876, 1999 Dorre, K., Bioimaging, 5: 139, 1997

  Therefore, the present inventors simultaneously have a feature as a support for immobilizing biomolecules and an adhesive for the surface of the chip substrate, so that it can be directly immobilized on the biochip without a separate apparatus and processing step. As a result of diligent efforts to develop an adhesive bead that can be used, and a biochip using the same, the present invention has been completed.

  An object of the present invention is to provide a support for fixing a biomolecule, an adhesive bead having characteristics as an adhesive to the surface of a chip substrate, and a method for producing the same.

  Another object of the present invention is to provide a biochip characterized in that a biomolecule is attached to the adhesive bead, and an aqueous suspension of the bead on which the biomolecule is immobilized is produced and then immobilized on a substrate. A manufacturing method and a biochip manufactured by the method are provided.

  To achieve the above object, the present invention provides a biomolecule produced by emulsifying a hydrophilic monomer, a main monomer, and a comonomer in an aqueous medium and then polymerizing the emulsion. An adhesive bead having characteristics as an adhesive to the support for fixing the substrate and the surface of the chip substrate is provided.

  In the present invention, the hydrophilic monomer is methacrylic acid, acrylic acid, itaconic acid, hydroxyethyl methacrylate, hydroxypropyl methacrylate, acrylamide, glycidyl methacrylate, polyethylene glycol acrylate, polyethylene glycol methacrylate, palitreic acid, oleic acid, linoleic acid, It may be characterized in that it is at least one selected from the group consisting of arachidonic acid, linolenic acid, allyl alcohol and vinyl alcohol, wherein the main monomer is butadiene, ethyl acrylate, butyl acrylate, ethyl hexyl acrylate and It may be characterized in that it is at least one selected from the group consisting of octyl acrylate, and the comonomer is vinyl acetate, acrylonitrile, acrylonitrile, Riruamido, styrene, can be characterized in that at least one selected from the group consisting of methyl methacrylate and methyl acrylate.

  The present invention also includes (a) adding a monomer to an aqueous emulsifier solution and mixing to make an emulsion, and (b) adding a hydrophilic monomer to the aqueous medium under a nitrogen atmosphere. After the temperature is raised to about 75 ° C., the emulsion obtained in the step (a) is added thereto, and the emulsion obtained by stirring is emulsified. (C) The emulsion obtained in the step (b) There is provided a method for producing an adhesive bead comprising a step of reacting after adding a polymerization initiator to a liquid.

  In the method for producing adhesive beads according to the present invention, the aqueous medium may be at least one selected from the group consisting of water, ethanol, methanol, DMF, DMSO, acetone, and NMP. The hydrophilic monomers are methacrylic acid, acrylic acid, itaconic acid, hydroxyethyl methacrylate, hydroxypropyl methacrylate, acrylamide, glycidyl methacrylate, polyethylene glycol acrylate, polyethylene glycol methacrylate, paroleic acid, oleic acid, linoleic acid, arachidonic acid, linolenic acid. It may be characterized by being at least one selected from the group consisting of acid, allyl alcohol and vinyl alcohol.

  In the method for producing adhesive beads according to the present invention, the monomer is at least one main monomer selected from the group consisting of butadiene, ethyl acrylate, butyl acrylate, ethyl hexyl acrylate, and octyl acrylate, vinyl acetate, acrylonitrile. , Acrylamide, styrene, methyl methacrylate, and at least one comonomer selected from the group consisting of methyl acrylate. The blending ratio of the main monomer and the comonomer is determined by the glass transition temperature (Tg) of the adhesive beads, and the glass transition temperature (Tg) is 0 to 0 based on the biochip production or use temperature. It can be characterized by being 45 ° C. lower.

  In the method for producing adhesive beads according to the present invention, the emulsifier is at least one selected from the group consisting of sodium lauryl sulfate, gelatin, methylcellulose, polyvinyl alcohol, cetyltrimethylammonium bromide, and sodium oleate. The polymerization initiator is at least one selected from the group consisting of potassium persulfate, ammonium persulfate, azobisbutyronitrile (AIBN), and benzoyl peroxide (BPO). can do.

  The present invention also includes (a) attaching a biomolecule to an adhesive bead and producing an aqueous suspension of the bead on which the biomolecule is immobilized; and (b) immobilizing the aqueous suspension on a substrate. The manufacturing method of a biochip including the process to make is provided.

  In the biochip manufacturing method according to the present invention, the step (b) is characterized in that after the aqueous suspension is spotted on the substrate, it is dried and fixed on the substrate. The spotting may be performed by inkjet, and a method of attaching a biomolecule to the adhesive bead is selected from the group consisting of a hydrophobic adsorption method, a covalent bond method, and a binding method by electrostatic attraction. It can be characterized by being any one selected.

  In the biochip manufacturing method according to the present invention, the biomolecule is any one selected from the group consisting of nucleic acids, amino acids, proteins, peptides, lipids, carbohydrates, enzyme substrates, ligands, and cofactors. The substrate may be any one selected from the group consisting of a microwell, a slide substrate, and a lab-on-chip fine flow path. The material is at least one selected from the group consisting of polymethyl methacrylate, polycarbonate, polystyrene, cyclic olefin copolymer, polynorbornene, styrene-butadiene copolymer, acrylonitrile butadiene styrene, glass, silicon, hydrogel, metal, ceramic, and porous membrane. Characterized by being one Rukoto can.

  The present invention also provides a biochip manufactured by the above method and having adhesive beads to which biomolecules are attached fixed to a substrate.

  The present invention also includes (a) applying a sample containing a target substance to a biochip, and (b) detecting a target substance that specifically binds to a biomolecule on the biochip. A method for detecting a target substance in a sample is provided.

  For the detection of the target substance, a detection method using a biolabel such as a radioisotope, luminescence or a coloring dye (biolabel), an enzyme immunoassay using a biological enzyme (enzyme-linked immunoassay, ELISA), Use at least one selected from the group consisting of electrochemical immunoassay, particle turbidimetric immunoassay, and detection using fluorophore Can be characterized.

  The present invention also provides a biochip for confirming the presence or absence of lamivudine-resistant hepatitis B virus infection, in which adhesive beads having SNP (single nucleotide polymorphism) of SEQ ID NO: 1 or SEQ ID NO: 2 are immobilized on a substrate I will provide a.

  The present invention also provides an uneven structure in which adhesive beads are coated on the surface of a chip substrate entirely or locally.

  Still other features and embodiments of the present invention will become more apparent from the following detailed description and appended claims.

  The description of FIGS. 1 to 17 will be described below.

  The present invention relates to a support for immobilizing a biomolecule, an adhesive bead having characteristics as an adhesive to the surface of the chip substrate, a manufacturing method thereof, and a bead having a biomolecule attached to the adhesive bead on the substrate. The present invention relates to a biochip that is fixed and a manufacturing method thereof. Below, the manufacturing method of the biochip by this invention is demonstrated according to process.

(First step: Production of aqueous suspension containing sticky beads)
The sticky beads of the present invention refer to a solid content having sticky properties in an aqueous suspension, a main monomer having stickiness, a comonomer having solidity, and hydrophilicity for aqueous dispersion Monomers are the basic constituents.

  The adhesive beads of the present invention are prepared by mixing a main monomer, a comonomer, and a hydrophilic monomer in an aqueous medium, and performing usual polymerization methods such as suspension, emulsification, dispersion, microemulsification, miniemulsion, and inverse emulsion polymerization. And beads of various sizes can be obtained according to the polymerization conditions. In order to use it as a support for immobilizing biomolecules, it is usually produced to a diameter of several tens of nanometers to several microns.

  Further, it is possible to simultaneously give the beads two functions as a support and an adhesive by adjusting the mixing ratio of the main monomer and the comonomer constituting the beads. Specifically, it is possible to select a copolymerization ratio that can simultaneously appear in the use environment of the biochip by using the soft and sticky adhesive property of the main monomer and the solid property of the comonomer. At this time, the most important factor for determining the mixing ratio of the main monomer and the comonomer is the inherent glass transition temperature (Tg) of the manufactured beads, and the Tg is the production or use of the biochip. It is preferably 0 to 45 ° C. lower than the temperature. For example, when a biochip is produced or used at room temperature (25 ° C.), the Tg of the adhesive beads is preferably in the range of −15 to 25 ° C., which is 0 to 45 ° C. lower than room temperature, and is −15 to 10 ° C. A range is further preferred.

(Second step: immobilization of biomolecules on adhesive beads and production of aqueous bead suspension containing the same)
The biochip of the present invention utilizes adhesive beads having a large surface area as a mediator in order to increase the accumulation rate of biomolecules immobilized on the chip substrate.

  Examples of methods for immobilizing biomolecules on beads include a hydrophobic adsorption method that interacts with the surface of the hydrophobic bead itself, a covalent bonding method that uses a specific reactive group of the copolymer chain that constitutes the bead, and electrostatic force. There is a combination method. As the aqueous medium used when producing the aqueous suspension of beads, any solvent having aqueous properties can be used. That is, the aqueous medium includes water, ethanol, methanol, DMF, DMSO, acetone, and NMP, but is not limited thereto, and water can be preferably used.

(3rd step: spotting of aqueous suspension of beads)
The bead suspension on which the biomolecules are immobilized can be immobilized on the surface of the substrate by spotting the suspension on the substrate. For the spotting, any spotting method usually used in the art can be used, and typically, a spotting method by ink jet can be used. When using the inkjet method, it is advantageous because the aqueous bead suspension according to the present invention can be easily ejected quantitatively onto the substrate.

  Various substrates used in the biochip field can be used as the substrate for fixing the adhesive beads. Typical examples include microwells, slide substrates, and lab-on-chip microchannels. It is not limited. Examples of the material for the substrate include polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), cyclic olefin copolymer, polynorbornene, styrene-butadiene copolymer (SBC), acrylonitrile butadiene styrene, glass, silicon, hydrogel, Metals, ceramics, and porous membranes can be used, but are not limited thereto.

(4th step: drying)
For drying, a normal drying method used when manufacturing a biochip by spotting can be used, for example, room temperature drying. Depending on the substance, there is an appropriate drying temperature, which is 15 to 33 ° C. for proteins and 15 to 90 ° C. for DNA.

  As a result of observing the biochip manufactured through the above process with a scanning electron microscope, as shown in FIG. 1 and FIG. 2, it is confirmed that the beads appearing through the fixing process bind to the substrate. it can. From the results, it was found that biochips can be produced using the adhesive beads according to the present invention.

(Application of biochip manufactured by the present invention)
The present invention includes a step of spotting and fixing an adhesive bead on which a biomolecule is immobilized on the chip substrate, applying a target substance sample to be detected, and a target substance specifically bound to the biomolecule And detecting the presence and content of the target substance present in the detection sample.

  In addition, the adhesive beads according to the present invention can be used to produce an uneven structure composed of beads by coating the surface of the chip substrate with the entire surface or locally (FIG. 7). Such a three-dimensional structure is used as a surface of a multi-area substrate for applying various useful functions in a biochip, for example, spotting and fixing biomolecules directly to a detection unit, or for a capillary. It can be used as a fluid delay part by a hydrophobic action by being applied to a specific fine flow path of a lab-on-chip that uses a flow.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, these Examples are only for demonstrating this invention. It should be apparent to those skilled in the art that the scope of the present invention should not be construed as limited to these examples.

Example 1: Production of aqueous suspension containing sticky beads
The main reactor was charged with 622.1 g of deionized water and 3.5 g of itaconic acid and heated to 75 ° C. under a nitrogen atmosphere. An emulsion prepared by mixing 35.0 g of butyl acrylate, 31.4 g of methyl methacrylate, 0.1 g of allyl methacrylate and 1.2 g of a 3 wt% aqueous sodium lauryl sulfate solution in a separate reactor was prepared.

  When the temperature of the main reactor is stabilized, the emulsion prepared in the separate reactor is added, and stirred for 1 hour or more to make it sufficiently emulsified. Then, 7.0 g of 3 wt% aqueous potassium sulfate solution is added twice. And 1.0 g were added and reacted for 2 hours to synthesize a polymer containing sticky beads.

  The polymer was washed with dialysis and ion exchange resin, and then diluted with deionized water to prepare an aqueous suspension containing sticky beads. The size of the adhesive beads can be adjusted by adjusting the amount of the emulsifier added. FIG. 3 shows the results of particle size analysis of beads produced with various amounts of emulsifier sodium lauryl sulfate, and when sub-emulsifiers in the range of 0.1 to 0.05% by weight are added, the average sub-micro size is shown. It has been found that a level of beads is produced.

  On the other hand, the glass transition temperature of the adhesive bead, which is a copolymer, is generally intermediate between the glass transition temperature of polybutyl acrylate, which is the main monomer polymer, and polymethyl methacrylate, which is the comonomer polymer. Indicates the value corresponding to the value. Therefore, by adjusting the blending ratio of each monomer, it was possible to produce adhesive beads having a target glass transition temperature (FIG. 4).

  FIG. 5 is a photograph of beads having different glass transition temperatures (Tg) observed with a scanning electron microscope. Referring to FIG. 5, beads having a low glass transition temperature under normal temperature drying conditions are strongly sticky and easily stick to a substrate, but are formed into a film and do not have a three-dimensional structure. The high bead had a strong solidity and a clear bead shape, but it was fragile and tended not to adhere well to the substrate. Therefore, it was found that adhesive beads having a glass transition temperature in the range of −15 to 10 ° C. are suitable when drying at room temperature.

(Example 2: Immobilization efficiency test by concentration of biomolecule)
Since the surface of the bead manufactured in Example 1 has both a surface to which the biomolecule is fixed and a surface to be adhered to the substrate, the area occupied by the biomolecule in the surface area of the entire bead is adjusted. The use efficiency of the adhesive beads that are the object of the present invention can be adjusted.

  As an immobilized biomolecule, an aqueous solution of bovine serum albumin in phosphate buffer (pH 7.2) was prepared at concentrations of 0.16, 0.31, 0.93, 1.55, and 3.10 mg / mL. Thereafter, the bead was mixed with an aqueous suspension containing 2% by weight of the beads at a ratio of 1: 1 and reacted by shaking at room temperature for 14 hours. After completion of the reaction, the supernatant was collected by centrifugation, the amount of bovine serum albumin not immobilized was measured, and the amount of bovine serum albumin immobilized on the beads was quantified (FIG. 6). As a result, as shown in FIG. 6, the coverage of the bead surface could be adjusted by adjusting the reaction amount of the immobilized biomolecule.

(Comparative Example 1: Measurement of fixing ratio by fixing time and comparison test with polystyrene beads)
The amount of biomolecule immobilized on the beads was measured according to the reaction time, and compared with the case of polystyrene beads, which are typical commercially available beads.

  An aqueous suspension containing 1.6 mg / mL of an aqueous solution of bovine serum albumin in phosphate buffer (pH 7.2) and 2% by weight of adhesive beads (diameter 510 nm) or polystyrene beads (diameter 600 nm) prepared in Example 1 The immobilization process was performed in the same manner as in Example 2. The surface coverage was measured according to the reaction time for immobilizing biomolecules on the beads (FIG. 7). As a result, as shown in FIG. 7, in the case of the adhesive beads produced in Example 1, the surface coverage of biomolecules can be adjusted by adjusting the reaction time, and commercialized polystyrene. It was found that it showed excellent immobilization efficiency equivalent to beads.

(Example 3: Spot shape depending on concentration of adhesive beads)
In the production of biochips using sticky beads, the shape of the spots formed on the substrate is affected by the bead content of the dispensed aqueous bead suspension.

  Aqueous beads suspensions 0.05, 0.1, 0.5, and 1% by weight containing the sticky beads (average diameter 510 nm, Tg-8 ° C.) prepared in Example 1 were prepared, and polymethyl methacrylate was prepared. 0.5 mL each was spotted on the substrate. After the substrate was dried at room temperature for 12 hours, the surface shape of each spot was observed (FIG. 8). As a result, as shown in FIG. 8, it can be seen that the higher the content of beads in the spot, the higher the density of the fixed beads, but when the content exceeds 0.5% by weight, the beads are fixed as a multilayer. It was found that film formation was performed by the behavior of polymer chains.

(Example 4: Formation of uneven structure by coating of adhesive beads)
An aqueous bead suspension containing 8.5% by weight of the sticky beads (diameter 510 nm, Tg-8 ° C.) produced in Example 1 was dip-coated on the surface of the polymethylmethacrylate substrate entirely or locally (FIG. 9). As a result, as shown in FIG. 9, it was confirmed that an uneven structure composed of single-layer beads was formed. Such an uneven structure can be used as a surface of a multi-area substrate or a fluid delay structure in a biochip.

(Example 5: Spot autofluorescence measurement and non-specific protein binding)
It is a preliminary experiment for applying the beads of the present invention to a biochip, in which an aqueous suspension of beads is nano-spotted on a chip substrate, and autofluorescence and non-specific binding (non-specific binding) The degree of was measured.

  3. 200 μL of bovine serum albumin or bovine serum albumin phosphate solution labeled with SAH (S-adenosyl-L-homocysteine) at a concentration of 1 mg / mL, and 2% by weight adhesive bead aqueous suspension prepared in Example 1 The suspension was mixed with 200 μL and reacted by shaking at room temperature for 15 hours. After completion of the reaction, washing was carried out through a centrifugal separation process, and then suspensions having a total bead content of 0.2 and 0.4% by weight were prepared. The bead aqueous suspension was spotted on a polymethyl methacrylate (PMMA) substrate in a volume of 50 nL using an inkjet layer, and the autofluorescence intensity of the fixed spot was measured using a fluorescent image scanner (Exxon). (FIG. 10). As a result of the quantification, as shown in FIG. 10, it was found that the autofluorescence intensity of the sticky bead spot was an SNR (signal to noise ratio) not exceeding 3.

  On the other hand, beads coated with bovine serum albumin spotted by the method described above were treated with an anti-SAH antibody aqueous solution to quantify the degree of nonspecific binding of the protein (FIG. 11). As shown in the quantification results of FIG. 11, the fluorescence intensity of the spot measured after the non-specific binding reaction of the anti-SAH antibody is an SN ratio not exceeding 3, so there is almost no non-specific binding of protein. I understood.

  The experimental result that the autofluorescence intensity and non-specific binding are insignificant means that the bead does not interfere with the fluorescence detection of the target substance during the reaction between the beads of the present invention and the target substance. It has been found that the bead support of the present invention can be suitably used for biochips.

(Example 6: Competitive immunodetection against SAH target substance)
In order to fabricate a biochip capable of detecting a SAH target substance, bovine serum albumin and SAH-labeled bovine serum albumin were respectively coated on adhesive beads in the same manner as in Example 5, and phosphate buffer Was used as an aqueous medium to produce a 0.4 wt% bead aqueous suspension. The prepared aqueous bead suspension is spotted on a PMMA slide at a volume of 50 nL, dried at 30 ° C. for 30 minutes and at room temperature for 20 hours, and then contains 3% by weight bovine serum albumin and 0.05% by volume Tween. The resulting solution was blocked with a phosphate buffer (pH 7.4) for 30 minutes and washed. For fluorescence detection, a secondary antibody labeled with Cy3 is reacted with anti-SAH antibody for 30 minutes in advance, and then mixed with various concentrations of SAH, which is the target substance to be detected, to form a chip. Immune reaction was performed competitively with the spot.

  The fluorescent scanner photograph of the spot by SAH concentration and the detection result are shown in FIG. 12 and FIG. 13 (-●-), respectively. The fluorescence signal was expressed as a relative value with the signal when the target substance SAH was not added being 100. In the detection of SAH by competitive immunodetection of the chip manufactured in this example, the fluorescence signal is reduced by 90% or more compared to the case where the target substance SAH is not present, and the detection upper limit (highest detection limit) at this time is About 2-5 μM.

(Comparative Example 2: Comparison with two-dimensional biomolecule immobilization method)
In order to verify the superiority of the three-dimensional immobilization method using the adhesive beads of the present invention, the immunodetection performance was compared with a conventional two-dimensional immobilization method commercialized as a biomolecule immobilization method.

  As a representative chip capable of two-dimensional immobilization of biomolecules, Nunc's MaxiSorp chip was selected to test competitive immunodetection against the SAH target substance, and the results were compared with Example 6.

  After preparing bovine serum albumin and SAH (S-adenosyl-L-homocysteine) -labeled bovine serum albumin in a phosphate buffer solution containing 20% by volume of glycerol in a concentration of 0.5 mg / mL, MaxiSorp Spotted and dried in a humidity chamber for 20 hours. After washing the spotted chip, a competitive immune reaction was performed in the same manner as in Example 6. As a result, as shown in FIG. 13 (-■-), the maximum decrease value of the fluorescence signal by the competitive reaction was about 50%, the standard quantification range was 0.01 to 0.5 μM, and The detection level of the immobilization method using sticky beads is not greatly affected.

  The above results are due to the fact that the conventional two-dimensional immobilization method using the MaxiSorp chip has a low degree of biomolecule integration and a large steric hindrance to the surface of the chip. From this result, it was confirmed that the biochip using the adhesive beads according to the present invention was relatively superior to the conventional biochip.

(Example 7: Specific SNP detection)
Single nucleotide polymorphism which is one of DNA detection application fields by fixing an oligonucleotide sequence used to confirm the presence or absence of infection with hepatitis B virus (HBV) having lamivudine resistance, a therapeutic agent for hepatitis B, to the adhesive beads (SNP: single nucleotide polymorphism) was detected.

  The HBV having lamivudine resistance is obtained by mutating the YMDD motif of the viral polymerase, and generally there is a YIDD mutant in which Met552 is replaced with isoleucine. There is only one base difference between the normal sequence of SEQ ID NO: 1 expressing the YMDD motif and the mutant sequence of SEQ ID NO: 2 expressing the YIDD motif (Table 1).

  A normal probe complementary to the HBV polymerase gene sequence having a YMDD motif and a mutant probe complementary to the gene sequence of the YIDD mutant are immobilized on the surface of the chip using the adhesive beads, respectively, and then labeled with a fluorescent dye It was tested whether selective detection was possible with respect to the HBV polymerase gene sequence (target).

  In order to effectively immobilize each oligonucleotide probe to an adhesive bead, sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate ) Was used to coat bovine serum albumin, and this was coated onto sticky beads in a manner similar to Example 6.

  A gene chip was manufactured by nano-spotting the aqueous beads suspension at a concentration of 0.4% by weight on a plastic substrate. The chip was washed with deionized water for 3 minutes, treated with 80 μL blocking solution (20 × SSC 3 mL, formamide 1.35 mL, 1 wt% BSA 500 μL, deionized water 150 μL) at 40 ° C. for 30 minutes, and then a target sample 5 μL of (0 nM to 100 nM) was added and hybridized at 40 ° C. for 1 hour. In order to remove the target sequence having non-specific binding, it was washed with 2X SSC for 10 minutes, 0.2X SSC for 10 minutes, and analyzed with a fluorescence scanner. FIG. 14 is fluorescence scanner photographs in which SNP (single nucleotide polymorphism) of an oligonucleotide having a polymerase gene sequence of hepatitis B virus (HBV) was detected at various concentrations using the biochip according to this example. These are the graphs which show the fluorescence signal intensity which analyzed the fluorescence scanner photograph of FIG.

  As a result of analyzing the fluorescence signal intensity from the measured fluorescence scanner photograph, the normal probe complementary to the target sample, which is the target substance, has a discrimination ability about 4.1 times higher than that of the mutant probe having a different base. I was able to confirm that From this result, it was found that the biochip according to the present invention is useful for the purpose of detecting a gene such as SNP.

(Example 8: Direct immunodetection against protein target substance)
It was tested that immunodetection using the above-mentioned adhesive beads was possible even for a protein antibody that is not a low-molecular substance such as SAH exemplified in Example 6 as a target substance.

  BSA (bovine serum albumin) was used as a protein antigen. A simple biomaterial capable of detecting IgG (immunoglobulin) by producing adhesive beads coated with BSA (Calbiochem, antigen grade) in the same manner as in Example 6 and spotting on PMMA substrate. I made a chip. For surface blocking, treatment was performed at room temperature for 30 minutes with 30% human serum diluted with 1X PBS buffer aqueous solution. Anti-SAH IgG (polyclonal antibody, 50 μg / mL) is selected as a negative control group, and anti-BSA IgG (monoclonal antibody, 50 μg / mL) is selected as a positive control group, and allowed to react at room temperature for 45 minutes on the chip substrate. Then, anti-mouse-Cy3 (polyclonal, 10 μg / mL) labeled with a Cy3 fluorescent dye as a secondary antibody was reacted at room temperature for 20 minutes. After washing away the remaining non-specific antibody with 1X PBS buffer aqueous solution, the fluorescence signal was detected using a fluorescence scanner. FIG. 16 is a fluorescence scanner photograph in which immunoglobulin (IgG), which is a protein antigen, is detected by a direct immunodetection method using the biochip of the present invention, and FIG. 17 is a fluorescence photograph obtained by analyzing the fluorescence scanner photograph in FIG. It is the graph which showed signal intensity.

  As a result of analyzing the fluorescence signal intensity from the measured fluorescence scanner photograph, the positive control group specific for the target substance IgG was 7.7 (positive signal intensity / negative compared to the non-specific negative control group. It was confirmed that the signal intensity was detectable. From this result, it was found that the biochip according to the present invention is also useful for the purpose of detecting protein antigens.

  The specific contents of the present invention have been described in detail above. However, such a specific technique is only a preferred embodiment for those having ordinary knowledge in the art, and thus the present invention is not limited thereto. It will be clear that the range is not limited. Accordingly, the substantial scope of the present invention should be defined by the appended claims and their equivalents.

  The adhesive beads according to the present invention simultaneously have a feature as an adhesive for the support for fixing biomolecules and the surface of the chip substrate, and can be directly fixed on the biochip without a separate apparatus and treatment process. In addition, the adhesive beads of the present invention have an advantage that the coverage of the bead surface can be adjusted according to the reaction amount of the biomolecule to be immobilized and the reaction time for immobilizing the biomolecule to the bead. Furthermore, while exhibiting immobilization efficiency similar to that of existing commercial beads, the biomolecule integration rate is higher than conventional two-dimensional biomolecule immobilization methods, and the biochip is manufactured in a small size. Therefore, there is an effect that it is excellent in terms of economy.

It is the photograph which expanded and observed 10,000 times with the scanning electron microscope that the adhesive bead (600 nm) by this invention was fixed on the board | substrate. 2 is a photograph of the beads of FIG. It is a graph which shows that the magnitude | size of a bead changes according to the content of the emulsifier input. It is a graph which shows that the glass transition temperature (Tg) of the bead manufactured changes according to the copolymerization ratio of a main monomer and a comonomer. It is the photograph which observed the bead which has a mutually different glass transition temperature with the scanning electron microscope. It is a graph which shows that the surface coverage with respect to the bead of a biomolecule increases so that the density | concentration of a biomolecule is high. It is a graph which shows that the surface coverage with respect to the adhesive bead and polystyrene bead of this invention of a biomolecule increases according to the reaction time which fixes a biomolecule to a bead. It is the photograph which observed with the scanning electron microscope that the bead aqueous suspension containing an adhesive bead was spotted on the polymethylmethacrylate board | substrate according to each weight%. An uneven structure formed by dip-coating a plastic substrate with an adhesive bead aqueous suspension was observed with a scanning electron microscope. It is the graph which measured and quantified the autofluorescence of the spot formed by spotting the aqueous | water-based suspension of the adhesive bead which fixed protein on the chip | tip substrate. It is the graph which processed nonspecific protein to the spot formed by spotting the aqueous suspension of the adhesive bead which fixed protein on the chip substrate, and quantified the degree of binding. It is the fluorescence scanner photograph detected by competitive immunodetection method with various concentrations of SAH (S-adenosyl-L-homocysteine) using the biochip of the present invention. It is a graph which shows the result of having performed the competitive immunity detection with respect to the target substance SAH using the biochip of this invention and the MaxiSorp chip | tip of Nunc. It is the fluorescence scanner photograph which detected SNP (single nucleotide polymorphism) of the oligonucleotide which has the polymerase gene sequence of hepatitis B virus (HBV) using the biochip of this invention in various density | concentrations. It is a graph which shows the fluorescence signal intensity | strength which analyzed the fluorescence scanner photograph of FIG. It is the fluorescence scanner photograph which detected the immunoglobulin (IgG) which is a protein antigen with the direct immunodetection method using the biochip of this invention. It is a graph which shows the fluorescence signal intensity | strength which analyzed the fluorescence scanner photograph of FIG.

Claims (23)

  The hydrophilic monomer, main monomer, and comonomer in an aqueous medium are emulsified and then polymerized. At the same time, the support for fixing biomolecules and the feature as an adhesive to the surface of the chip substrate are simultaneously used. Sticky beads with.   The hydrophilic monomer is methacrylic acid, acrylic acid, itaconic acid, hydroxyethyl methacrylate, hydroxypropyl methacrylate, acrylamide, glycidyl methacrylate, polyethylene glycol acrylate, polyethylene glycol methacrylate, paroleic acid, oleic acid, linoleic acid, arachidonic acid, linolenic acid. The adhesive bead according to claim 1, wherein the adhesive bead is at least one selected from the group consisting of acid, allyl alcohol and vinyl alcohol.   The adhesive bead according to claim 1, wherein the main monomer is at least one selected from the group consisting of butadiene, ethyl acrylate, butyl acrylate, ethyl hexyl acrylate, and octyl acrylate.   The adhesive bead according to claim 1, wherein the comonomer is at least one selected from the group consisting of vinyl acetate, acrylonitrile, acrylamide, styrene, methyl methacrylate, and methyl acrylate. A method for producing an adhesive bead comprising the following steps:
(A) a step of adding a monomer to an aqueous emulsifier solution and mixing to make an emulsion;
(B) A hydrophilic monomer is added to the aqueous medium, the temperature is raised to about 75 ° C. under a nitrogen atmosphere, and then the emulsion obtained in the step (a) is added thereto and stirred to make the emulsion emulsified. And (c) a step of adding a polymerization initiator to the emulsion obtained in the step (b) and then reacting the emulsion.   The method according to claim 5, wherein the aqueous medium is at least one selected from the group consisting of water, ethanol, methanol, DMF, DMSO, acetone and NMP.   The hydrophilic monomer is methacrylic acid, acrylic acid, itaconic acid, hydroxyethyl methacrylate, hydroxypropyl methacrylate, acrylamide, glycidyl methacrylate, polyethylene glycol acrylate, polyethylene glycol methacrylate, paroleic acid, oleic acid, linoleic acid, arachidonic acid, linolenic acid. The method according to claim 5, wherein the method is at least one selected from the group consisting of acid, allyl alcohol and vinyl alcohol.   The monomer is at least one main monomer selected from the group consisting of butadiene, ethyl acrylate, butyl acrylate, ethyl hexyl acrylate and octyl acrylate, and vinyl acetate, acrylonitrile, acrylamide, styrene, methyl methacrylate and methyl acrylate. The method according to claim 5, comprising at least one comonomer selected from the group consisting of:   The method according to claim 5, wherein the emulsifier is at least one selected from the group consisting of sodium lauryl sulfate, gelatin, methylcellulose, polyvinyl alcohol, cetyltrimethylammonium bromide, and sodium oleate.   6. The polymerization initiator according to claim 5, wherein the polymerization initiator is at least one selected from the group consisting of potassium persulfate, ammonium persulfate, azobisbutyronitrile (AIBN), and benzoyl peroxide (BPO). The method described.   The mixing ratio of the main monomer and the comonomer is determined by the glass transition temperature (Tg) of the adhesive beads, and the glass transition temperature (Tg) is 0 to 45 from the production or use temperature of the biochip. The method according to claim 8, wherein the method is low. Biochip manufacturing method including the following steps:
(A) a step of producing an aqueous suspension of beads having biomolecules immobilized thereon by attaching biomolecules to the adhesive beads according to any one of claims 1 to 4; and (b) the aqueous solution. Fixing the suspension on the substrate.   The method according to claim 12, wherein the step (b) comprises spotting the aqueous suspension on the substrate, and then drying and fixing the aqueous suspension on the substrate.   The method according to claim 12, wherein the spotting is performed by inkjet.   The method for attaching a biomolecule to the adhesive bead is any one selected from the group consisting of a hydrophobic adsorption method, a covalent bonding method, and a binding method by electrostatic attraction. The method described in 1.   The method according to claim 12, wherein the biomolecule is any one selected from the group consisting of nucleic acids, amino acids, proteins, peptides, lipids, carbohydrates, enzyme substrates, ligands and cofactors. .   The method according to claim 12, wherein the substrate is one selected from the group consisting of a microwell, a slide substrate, and a lab-on-chip microchannel.   The substrate material is selected from the group consisting of polymethyl methacrylate, polycarbonate, polystyrene, cyclic olefin copolymer, polynorbornene, styrene-butadiene copolymer, acrylonitrile butadiene styrene, glass, silicon, hydrogel, metal, ceramic, and porous membrane. The method according to claim 17, wherein the method is at least one.   A biochip manufactured by the method according to claim 12, wherein adhesive beads to which biomolecules are attached are fixed to a substrate. A method for detecting a target substance in a sample, comprising the following steps:
(A) applying a sample containing a target substance to the biochip according to claim 19; and (b) detecting a target substance that specifically binds to a biomolecule on the biochip.   The detection of the target substance is performed using a detection method using a biolabel such as a radioisotope, luminescence, or coloring dye, an enzyme immunoassay (ELISA) using a bioenzyme, an electrochemical immunoassay, or a particle. 21. The method according to claim 20, wherein at least one selected from the group consisting of an immunoturbidimetric method and a detection method using a fluorescent chromophore is used.   A biochip for confirming the presence or absence of lamivudine-resistant hepatitis B virus, wherein the beads are fixed on a substrate, wherein the beads are arrayed on the adhesive beads according to any one of claims 1 to 4. A biochip with SNP (single nucleotide polymorphism) of No. 1 or SEQ ID No. 2.   An uneven structure in which the adhesive beads according to any one of claims 1 to 4 are entirely or locally coated on the surface of a chip substrate.

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